OBJECTIVE: To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. METHODS: The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. RESULTS: The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. CONCLUSIONS We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.
Animals ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Receptor, ErbB-3 ; immunology ; Recombinant Fusion Proteins

Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
Acrosome ; immunology ; Animals ; Antibodies ; analysis ; Blotting, Western ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; immunology ; Escherichia coli ; Humans ; Immunohistochemistry ; Male ; Muramidase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; Semen ; immunology ; Spermatozoa ; immunology ; Testis ; immunology

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

Listeria monocytogenes (L. monocytogenes, LM) is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16 (HPV16) E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly::E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly::E7 could express and secrete E7-LLO fusion protein; its size is 66 kDa and has immunological activity. Furthermore, LM4△hly::E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly::E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose (LD₅₀) of LM4△hly::E7 strain was 3.863×10⁹ CFU (Colony-Forming Units) in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly::E7 did not show obvious pathological change. These data show that LM4△hly::E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.
Animals ; Cancer Vaccines ; immunology ; Listeria monocytogenes ; Mice ; Mice, Inbred C57BL ; Papillomavirus E7 Proteins ; immunology ; Papillomavirus Infections ; prevention & control ; Plasmids ; RAW 264.7 Cells ; Recombinant Fusion Proteins ; immunology ; Vaccines, Attenuated ; immunology ; Viral Vaccines ; immunology

OBJECTIVETo construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.

METHODSLuciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.

RESULTSThe shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.

CONCLUSIONChemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.

Adenoviruses, Human ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Callithrix ; Cell Line ; Humans ; Immunity, Humoral ; Luciferases ; Plasmids

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.
Algal Proteins ; biosynthesis ; immunology ; Animals ; Antibodies ; chemistry ; Blotting, Western ; Chlamydomonas reinhardtii ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Fluorescent Antibody Technique ; Intracellular Signaling Peptides and Proteins ; biosynthesis ; immunology ; Plasmids ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis

OBJECTIVETo construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway.

METHODSThe nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay.

RESULTSThe recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1.

CONCLUSIONWe successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Allergens ; immunology ; Animals ; Antigens, Dermatophagoides ; immunology ; Arthropod Proteins ; immunology ; Base Sequence ; Cloning, Molecular ; Cysteine Endopeptidases ; immunology ; Dermatophagoides pteronyssinus ; Epitopes, T-Lymphocyte ; Escherichia coli ; Gene Expression ; Genes, MHC Class II ; Genetic Vectors ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Vaccines ; immunology

To increase detection sensitivity and specificity on hepatitis C virus (HCV) is vital for prevention and controlling of the disease. To establish a more reliable detection method for HCV diagnosis, the full gene fragment of ns3 (non-structural protein of HCV) from recombinant plasmid of J6/JFH1 2a was amplified and then connected into the pET-28a prokaryotic expression vector, and the latter was subsequently transformed into Escherichia coli BL21 (DE3) to have the target protein expression. As a result, a protein with a molecular weight of 72 kDa was obtained and visualized in 10% SDS-PAGE. The purified NS3 protein was used as immunogen to inoculate BALB/c mice and the sera was collected after the fourth immunization. The antibody titer of serum is determined to be about 1:256000 with ELISA. Western blotting and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native NS3 protein in Huh 7.5.1 cells infected with HCV. These findings may provide basis for further preparation of monoclonal antibodies against NS3 and the development of related detection kit.
Animals ; Antibodies, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Hepacivirus ; Mice ; Mice, Inbred BALB C ; Plasmids ; Viral Nonstructural Proteins ; biosynthesis ; immunology

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

OBJECTIVETo investigate the humoral and cellular immune responses induced by MUC1-2VNTR DNA vaccine in multiple myeloma (MM) tumor-bearing mice.

METHODSIn vitro, multiple myeloma cells were transfected by plasmid pcDNA3.1-2VNTR/myc-hisB with Lipofectamine2000. The above-mentioned mouse myeloma cells were inoculated subcutaneously into female BALB/c mice for establishing tumor-bearing animal models. These female BALB/c mice were immunized with pcDNA-2VNTR/myc-hisB or pcDNA/myc-hisB. The cytotoxic T lymphocyte (CTL) activity was detected by the LDH method and the spleen lymphocyte proliferation activity was detected by CCK-8 method.

RESULTSAfter immunization of BALB/c tumor-bearing mice with recombinant plasmid for 25 days, the tumor mass (0.5605 ± 0.2065 g) was significantly lighter than that in the empty plasmid control group (1.521 ± 0.6985 g) (P < 0.01) and the control group (1.5315 ± 0.5425 g) (P < 0.01). The difference of tumor mass was not statislically significant between empty plasmid control group (1.521 ± 0.6985 g) and the control group (1.5315 ± 0.5425 g) (P > 0.05). The CTL and NK cell activity was significantly higher in the group of intramuscular injection with recombinant plasmid than that in control group. The spleen lymphocyte proliferation was statistically significantly increased after being immunized with recombinant plasmid pcDNA3.1-2VNTR/myc-hisB, compared with empty vector (P < 0.01). The results showed that MUC1-2VNTR gene immunization could induce anti-tumor effect in MM tumor-bearing mice.

CONCLUSIONMUC1-2VNTR DNA immunization can elicit both humoral and cellular tumor specific immune response to multiple myeloma in MM tumor-bearing mice. It suggested that the MUC1-2VNTR DNA vaccine may be a potential treatment measure for patients with MM.

Animals ; Cancer Vaccines ; therapeutic use ; Female ; Genetic Vectors ; Humans ; Immunization ; Killer Cells, Natural ; immunology ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Minisatellite Repeats ; Mucin-2 ; genetics ; Multiple Myeloma ; immunology ; therapy ; Neoplasm Transplantation ; Plasmids ; Spleen ; cytology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, DNA ; therapeutic use